Original Article P-Glycoprotein, Multidrug Resistance-Associated

Antiviral Therapy 2009 14: 965–974 (doi: 10.3851/IMP1399)

Original article P-glycoprotein, multidrug resistance-associated proteins and human organic anion transporting polypeptide influence the intracellular accumulation of atazanavir Omar Janneh1,2*, Tariq Anwar3, Christof Jungbauer 4, Stefan Kopp5, Saye H Khoo2, David J Back2 and Peter Chiba5

1Department of Biomolecular and Sport Sciences, Coventry University, Coventry, UK 2Department of Pharmacology, School of Biomedical Sciences, University of Liverpool, Liverpool, UK 3School of Biomedical Sciences, University of Ulster, County Londonderry, UK 4Austrian Red Cross, Blood Donation Centre for Vienna, Lower Austria and Burgenland, Vienna, Austria 5Institute of Medical Chemistry, University of Vienna, Vienna, Austria

*Corresponding author: e-mail: [email protected]

Background: Drug efflux (for example, P-glycoprotein HPIs on uptake were also evaluated. Data from digitonin [P-gp], multidrug resistance-associated proteins [MRPs] permeabilized cells allowed the evaluation of the contri- and breast cancer resistance protein [BCRP]) and influx bution of cellular binding to total drug uptake, whereas (for example, human organic anion transporting polypep- the inhibitory effect of ATV on P-gp was assessed by tide [hOCTP] or human organic anion transporting daunomycin efflux/uptake assays. polypeptide [hOATP]) transporters alter the cellular con- Results: [3H]-ATV is lipophilic and accumulates in the

centrations of some HIV protease inhibitors (HPIs). Here, cultured cells as follows: CEM>CEME1000>CEMVBL. Tariq- we studied the lipophilicity and uptake of [3H]-atazanavir uidar, GF120918 and daidzein significantly increased the 3 (ATV) in CEM (parental), CEMVBL (P-gp-overexpressing), uptake of [ H]-ATV in the cultured cells. By contrast, only

CEME1000 (MRP1-overexpressing) and peripheral blood daidzein and tipranavir significantly increased uptake in mononuclear cells (PBMCs), and evaluate the effects of PBMCs, with tariquidar and frusemide devoid of effects, modulators of drug transporters on uptake. whereas dipyridamole, E-3-S, GPV031 and genistein sig- Methods: Lipophilicity was measured by octanol/saline nificantly decreased accumulation. ATV inhibits P-gp partition method. The influence of influx/efflux trans- activity; manipulation of uptake with digitonin suggests porters on uptake was evaluated in the absence and binding of [3H]-ATV to P-gp. presence of inhibitors of P-gp (GPV031), P-gp/BCRP Conclusions: [3H]-ATV is lipophilic, a P-gp, MRP and (tariquidar and GF120918), P-gp/MRP1 (dipyrida- hOATP substrate and an inhibitor of P-gp. Concomitant mole and daidzein), MRP1/2 (frusemide and genistein), administration of ATV with drugs and dietary components hOATP/hOCTP (estrone-3-sulfate [E-3-S]) and hOATP/ (for example, daidzein and genistein) that interact with hOCTP/MRP (probenecid). The effects of a number of these transporters could alter its pharmacokinetics.

Introduction

HIV protease inhibitors (HPIs) in combination with resistance-associated proteins (MRPs) and breast can- other antiretrovirals (for example, reverse tran- cer resistance protein (BCRP) [1–3]. In addition, there scriptase inhibitors) play a pivotal role in the man- are some known anionic and cationic transporters, agement of HIV-infected patients. The therapeutic such as human organic anion transporting polypep- effects of these drugs is dependent on their cellular tide (hOATP) and human organic cation transport- accumulation, which is in part affected by the expres- ers (hOCTs), respectively, which mediate the influx/ sion of ATP binding cassette family of drug efflux elimination of many clinically important drugs and transporters, such as P-glycoprotein (P-gp), multidrug xenobiotics, and these have been shown to limit the

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accumulation of some HPIs [2,4–6]. All these trans- Methods porters are differentially expressed in the liver, kidney, circulating lymphocytes, brain, testes and intestines – Reagents sites important in drug disposition, or which might act [3H]-ATV and [14C]-mannitol (specific activities 3.1 Ci/ as sanctuary for the replicating virus and/or support mmol and 50 mCi/mmol, respectively) were purchased viral replication. Clearly, low intracellular/plasma from Moravek Biochemicals and Radiochemicals (Brea, drug concentration or altered distribution within these CA, USA). RTV and lopinavir (LPV) were obtained sites could affect clinical outcome. from Abbott Laboratories (North Chicago, IL, USA); The transporters work in concert to control the bio- nelfinavir (NFV), indinavir (IDV), SQV, amprenavir availability of clinically important drugs and endog- (APV), ATV and tipranavir (TPV) were obtained from enous compounds. Thus, together with the cytochrome Agouron Pharmaceuticals (San Diego, CA, USA), Merck P450 enzymes, these transporters are important deter- (Rahway, NJ, USA), Roche (Welwyn Garden City, UK), minants of drug–drug interactions, drug efficacy and Glaxo Wellcome (Middlesex, UK), Bristol–Myers Squibb toxicity [7]. Indeed, previous studies have shown (Hounslow, UK) and Boehringer Ingelheim (Berkshire, the oral availability, brain, and fetal penetration of UK), respectively. Tariquidar was donated by Xenova saquinavir (SQV) to be limited by P-gp [8,9]. Simi- Group Plc (Berkshire, UK). The peripheral blood mono- larly, the intracellular accumulations of a number of nuclear cells (PBMCs) were isolated from blood buffy HPIs have been shown to be limited by P-gp, MRP1, coats, which were obtained from the Regional Blood MRP2 and BCRP [1,2,8–19]. There is ample evidence Transfusion Centre (Liverpool, UK). CEM (parental),

that some HPIs also induce and inhibit the expression CEMVBL (VBL) and CEME1000 (E1000) cell lines were of drug efflux transporters, such as P-gp, MRP1/2 and from R Davey (Bill Walsh Cancer Research Laborato- BCRP [12,16,20–25]. However, the role of BCRP in ries, Royal North Shore Hospital, Sydney, Australia). the efflux of HPIs is equivocal [1,3,24–27]; there is Lymphoprep was purchased from Axis Shields (Oslo, evidence that BCRP induces resistance to nucleoside Norway). GPV031 was a gift from G Ecker (Depart- reverse transcriptase inhibitors [27]. ment of Medicinal Chemistry, University of Vienna, We have shown that the intracellular concentrations Vienna, Austria). All other chemicals were supplied by of HPIs are amenable to manipulation by coadministra- Sigma Chemical Co. (Poole, UK). tion with inhibitors of drug efflux transporters [1,2,13]. A potential use of such agents would be to enhance intes- Cell culture tinal drug absorption and increase drug penetration to The cultured cells used in this study were CEM (a biologically important protective barriers, such as the CD4+ T-cell line), VBL (P-gp overexpressing; selected blood–brain, blood–cerebrospinal fluid, maternal–fetal using vinblastine) and E1000 (MRP-1 overexpress- barriers, testes, and within circulating lymphocytes ing; selected using epirubicin). The expression of these that might be impenetrable to these drugs. To this end, transporters has been previously validated in our lab- because ritonavir-mediated inhibition of cytochrome oratory [13]. The cells were maintained at 37°C, 5%

P450 (CYP3A4/CYP2D6) and possibly P-gp has been CO2 in RPMI-1640 media supplemented with 10% shown to increase exposure to coadministered HPIs, fetal bovine serum (FBS). ritonavir (RTV)-boosted HPIs are a standard compo- nent of antiretroviral regimen [28]. Interestingly, we and Isolation of PBMCs others have recently shown that other HPIs, such as ata- PBMCs were isolated from blood buffy coats using Lym- zanavir (ATV), can pharmacoenhance the intracellular phoprep according to the manufacturer’s instructions. accumulation and area under the curve of coadministed HPIs [2,29]. Published evidence to date suggests that Octanol/saline partition coefficient ATV might be a substrate, or induces or inhibits P-gp, As the cellular association of a drug within target cells MRP and BCRP activities [2,3,23,25,30,31], but does is a composite of active influx/efflux, passive diffusion not alter lymphocyte expression of P-gp, and MRP1 and ion trapping, the lipophilicity of [3H]-ATV was [3,31]. The current study aims to characterize the measured as described previously [6] to establish if sim- determinants of the cellular transport and sequestration ple physiochemical characteristics were major determi- of [3H]-ATV in cultured CD4+ T-cells and in primary nants of intracellular accumulation. human lymphocytes – an important site to study the transport and intracellular concentration of antiretro- Transport of [3H]-ATV and the effects of specific

virals. Experiments were designed to understand the inhibitors on transport in CEM and CEMVBL cells lipophilicity and sequestration of [3H]-ATV within cells Initial experiments examined the optimal concentrations and the efflux and influx pumps relevant for the vecto- of inhibitors for maximal inhibitory effects on Pgp, MRP rial transport and retention of the drug. and hOATP activity. Cells (CEM and its variant cells)

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were incubated in the absence or presence of varying incubating the incubation mixture with 20 µM digitonin concentrations of XR9576 (0.01–1 µM; P-gp/BCRP for 5 min once steady-state was reached (15 min). inhibitor [32–34]), MK571, dipyridamole (1–100 µM; P-gp/MRP inhibitor [35,36]), frusemide (1–100 µM; Effects of ATV on daunomycin accumulation and MRP1/2 inhibitor [1], GF120918 (1–100 µM; P-gp/ efflux in cultured cells BCRP inhibitor [13,37]), GPV031 (1–100 µM; P-gp Daunomycin is a substrate of P-gp and its accumulation inhibitor [38]), probenecid (1–100 µM; MRP/hOATP and efflux were performed as described previously [38]. [14,39] and E-3-S (1–100 µM; hOATP/hOCTP inhibi- Concentration–response curves were fitted to the data tor [6,40]) as described [1]. points using non-linear least-squares and 50% effective In subsequent experiments, the transport and accu- concentration values were calculated as described pre- mulation of [3H]-ATV (20 nM) was measured by incu- viously [38]. bating the cells (2.5×106 cells/ml) in the absence and presence of fixed concentrations of inhibitors: tariqui- Statistical analyses dar (1 µM), frusemide (50 µM), dipyridamole (50 µM), Data are expressed as mean ±s d and the distribution of E-3-S (30 and 100 µM), probenecid (30 and 100 µM), data was assessed using the Shapiro–Wilk test. Statisti- GPV031 (30 and 100 µM) and GF120918 (50 µM). We cally significant differences between controls and drug- also investigated the interaction of soy-derived phytoes- treated samples were further assessed using the Mann– trogens daidzein and genistein (at 1 and 5 mM) known Whitney U test of the StatsDirect statistical software to interact with P-gp, MRP, BCRP and hOATP [41–47] (version 2.6.3; Cheshire, UK). Differences between on the cellular accumulation ratio (CAR) of [3H]-ATV. means were considered to be significant for P<0.05. The interaction of the HPIs, APV, IDV, LPV, NFV, RTV, SQV and TPV (all at 10 and 30 µM) on the transport Results and accumulation of [3H]-ATV was also investigated in CEM and its variant cells. All experiments were carried Octanol/saline partition coefficient out in RPMI-1640 media supplemented with 10% FBS The octanol/saline partition coefficient for [14C]- as described previously [1]. The amount of drug associ- mannitol (used as control) and [3H]-ATV were 0.002 ated with the cells was quantified by taking a ratio of ±0.001 and 29.90 ±4.12, respectively. the amount of drug in the cell pellets to the amount of drug in a similar volume of the bathing media after Transport of [3H]-ATV and the effects of inhibitors incubation, taking cell volume (1 pl) into consideration. of drug efflux/influx transporters and digitonin on This ratio was defined as the CAR. accumulation As can be seen in Figure 1A, we observed differential Accumulation of [3H]-ATV in isolated PBMCs and the accumulation of [3H]-ATV in the CEM, E1000 and VBL effects of modulators of accumulation cells with the baseline CAR of CEM, E1000 and VBL The assay was carried out essentially as described cells being 11.2 ±0.9, 8.4 ±0.9 and 7.0 ±0.6, respectively. above. Briefly, isolated PBMCs (5×106 cells/ml; cell vol- Tariquidar, dipyridamole and GPV031 (at 30 µM) signif- ume 0.4 pl) were incubated in the absence or presence icantly decreased (P≤0.05) the CAR of ATV in the CEM of fixed concentrations of the inhibitors: tariquidar cells. Similarly, tariquidar and GPV031 (at 30 µM), and (1 µM), GF120918 (50 µM), frusemide (50 µM), dipy- daidzein (at 1 mM), significantly (P≤0.05) decreased the ridamole (50 µM), GPV031 (30 and 100 µM), E-3-S CAR of [3H]-ATV in the E1000 cells. Genistein signifi- (30 and 100 µM), and APV, IDV, LPV, NFV, RTV, SQV cantly (P≤0.05) decreased the CAR of [3H]-ATV in the and TPV (all at 10 and 30 µM). In addition, we inves- CEM and its variant cells. Tariquidar and GF120918 tigated the interaction of daidzein and genistein (1 and significantly (P≤0.05) increased the CAR of [3H]-ATV 5 mM, respectively) on the CAR of [3H]-ATV. in the VBL cells, and daidzein (at 5 mM) increased the CAR of [3H]-ATV in CEM and its variant cells (P<0.05). Effects of digitonin permeabilization on the transport Frusemide, daidzein (at 1 mM) and genistein signifi- of [3H]-ATV cantly (P≤0.01) decreased the CAR of [3H]-ATV in the In order to characterize the energy dependence and accu- VBL cells. E-3-S (at 30 µM) reduced the CAR of [3H]- mulation of [3H]-ATV, drug transport assays were carried ATV in E1000 and VBL cells (P<0.05), with 100 µM out in cells pretreated without or with 20 µM digitonin as E-3-S causing a significant (P≤0.01) decrease in the CAR described previously [48] followed by steady-state drug of [3H]-ATV in CEM and VBL cells. To characterize the accumulation assay. Cells were incubated with 20 µM contribution of specific cellular binding to total the accu- digitonin prior to the addition of [3H]-ATV (20 nM), mulation of ATV, we investigated the effects of digitonin after which cells were incubated for a further 15 min. In permeabilization on accumulation. Here, cells were either other experiments, the cells were permeabilized by further initially permeabilized with 20 µM digitonin prior to the

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3 Figure 1. Effects of modulators of drug transporters, digitonin and HPIs on the accumulation of [ H]-atazanavir in CEM, CEMVBL, CEME1000 and PBMCs

A CEM

CEME1000 16 a CEMVBL

b 12

a a a a 10 a c a a

a 8 a a a a a a a CAR of ATV of CAR a a b a c 6 a a a c c 4

r S n d

Control riquida M E-3- M E-3-S Ta µ µ M GPV031M GPV031 M Digitonin M M FrusemideM GF120918 µ µ 30 µ µ µ µ 100 M ProbeneciM Probenecid 1 M Dipyridamole 30 1 mM Daidzein5 mM Daidzein1 mM Genistei5 mM Genistein µ µ 20 µ 100 50 50 30 50 100

B 12 CEM

CEME1000 c 10 CEMVBL

a 8 a

6 a CAR of ATV of CAR

4 a a

c c c c

0 V V V V V V V V V RT RT M IDV M IDV Control M AP M AP µ µ M LPV M LP M NF M NF M M M SQ M SQ µ µ µ µ µ µ µ µ µ µ 10 30 10 30 10 30 10 30 10 30 10 30

Effects of (A) modulators of drug transporters (tariquidar, dipyridamole, frusemide, GF120918, GPV031, daidzein, genistein, estrone-3-sulphate [E-3-S] and probenecid), digitonin and (B) HIV protease inhibitors (HPIs; amprenavir [APV], indinavir [IDV], lopinavir [LPV], nelfinavir [NFV], ritonavir [RTV] and saquinavir [SQV]) on the accumulation 3 of [ H]-ATV in CEM, CEME1000 and CEMVBL cells. (C) Modulators of drug transporters (tariquidar, frusemide, dipyridamole, E-3-S, GPV031, daidzein and genistein) and HPIs (APV, IDV, LPV, NFV, SQV and tipranavir [TPV]) on the accumulation of [3H]-ATV in isolated peripheral blood mononuclear cells (PBMCs) from blood buffy coats. Bars indicate mean ±s d (n=4, with 4 independent observations from cultured CEM and its variant cells and n=6, with 4 independent observations from each buffy coat PBMC sample). Results are expressed as cellular accumulation ratio (CAR), which is the ratio of the amount of [3H]-ATV associated with the cell pellets to the amount in a similar volume of media after incubation. P-values of aP<0.05, bP<0.001 and cP<0.01 indicate statistically significant differences (increases/decreases) in the CAR of [3H]-ATV as a result of coincubation with the compounds compared with control. Data were analysed by the Shapiro–Wilk test followed by the Kruskal–Wallis test.

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Figure 1. Continued

C a 30

a c a a 15

c a CAR of ATV

l V V

Contro 10 M30 IDV M IDV 10 µM30 APV µM APV µ µ 10 µM30 LPV µM LPV 10 µM30 NFV µM NFV 10 µM30 RT µM RT 10 µM30 SQV µM SQV 10 µM30 TPV µM TPV 30 µM E-3-S 100 M E-3-S 30µ M GPV031 1 mM5 DaidzeinmM Daidzein 1 µM Tariquidar 100µ M GPV031 1 mM5 mMGenistein Genistein 50 µM Frusemide µ 50 µM Dipyidamole

accumulation studies or subsequent to the accumulation cells. By contrast, LPV and RTV (at 30 µM) increased studies. We observed that permeabilization of CEM and the accumulation of [3H]-ATV in the E1000 cells. In E1000 cells with digitonin significantly (P<0.05) reduced the VBL cells, the accumulation of [3H]-ATV was sig- the accumulation of [3H]-ATV in these cells by 47% and nificantly (P<0.01) reduced by APV, IDV and RTV (at 30%, respectively. Unexpectedly, permeabilization of 30 µM). The uricosuric agent, probenecid (at 100 µM) VBL cells with digitonin caused a significant (P<0.01) significantly (P<0.05) increased the accumulation of increase in accumulation (from 7.0 ±0.6 to 15.9 ±2.6; [3H]-ATV in the E1000 cells, but had no effects on Figure 1A). Identical results were obtained in experi- accumulation in CEM and VBL cells (Figure 1B). ments in which the effect of digitonin was assessed subsequent to the accumulation studies (data not shown). Transport of [3H]-ATV in PBMCs and the effects of drug efflux inhibitors and HPIs on accumulation Effects of various HPIs on the accumulation of [3H]-ATV We observed variable accumulation of [3H]-ATV in RTV is used to boost the plasma concentration of a the PBMCs ranging from 13.6 ±1.5 to 24.9 ±1.9. number of HPIs. Here, we investigated whether RTV We previously showed that PBMCs express P-gp, and other HPIs could modulate the transport and accu- MRP, BCRP and hOATP [1,6]. Dipyridamole, E-3-S mulation of [3H]-ATV in the CEM and its variant cells. and GPV031 caused a modest decrease in accumula- Of the HPIs tested, only LPV (at 30 µM) significantly tion (Figure 1C). Interestingly, daidzein significantly (P<0.01) increased the association of [3H]-ATV to the increased (P<0.05), whereas genistein caused a sig- E1000 cells. We observed differential accumulation of nificant (P<0.01) decrease in the CAR of [3H]-ATV. [3H]-ATV in CEM, E1000 and VBL cells, with the fol- Unexpectedly, of all the HPIs tested, only TPV (at lowing rank order: CEM>E1000>VBL. LPV and NFV 30 µM) caused a modest (P<0.05) increase in the (at 30 and 10 µM, respectively) significantly (P<0.05) CAR of [3H]-ATV. APV, NFV, RTV and SQV caused decreased the accumulation of [3H]-ATV in the CEM a decrease (P<0.05) in the CAR of [3H]-ATV, either

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Figure 2. Effects of modulators of drug transporters and digitonin, alone and in combination, on the accumulation of [3H]-ATV in PBMCs

14 a b a b 12 a a 10

8 CAR of ATV 6

l e e r e e ro S S S S ont C M E-3- M E-3- M E-3-M E-3- µ µ µ µ M Digitonin M Tariquidar µ M tariquidaM frusemid µ M Frusemid µ µ 100 1 µ M Dipyidamol20 M dipyridamol 50 µ µ 50

M Digitonin; 1 M Tariquidar;M Frusemide; 100 100 µ M Digitonin; 50 µ µ µ 1 M Dipyridamole; 100 20 M Digitonin; 50 50 µ 20 µ 50 20

Data were tested for normality of distribution by the Shapiro–Wilk test followed by a parametric paired t-test. Each bar represents the mean ±s d (n=3, with 4 incubations per treatment). aP<0.01 and bP<0.05 compared with respective controls. ATV, atazanavir; CAR, cellular accumulation ratio; E-3-S, estrone-3-sulphate; PBMCs, peripheral blood mononuclear cells.

at both of the concentrations tested or at higher cells pretreated with digitonin followed by dipyridamole concentrations. over cells treated with digitonin alone. Identical results were obtained with cells that were initially pretreated Effects of digitonin permeabilization on the transport with the inhibitors and subsequently permeabilized with of [3H]-ATV digitonin (data not shown). We also investigated the Here, cells were either initially permeabilized with 20 µM effects of E-3-S (alone and in the presence of tariquidar-, digitonin prior to the accumulation studies (in the absence frusemide- or dipyridamole-treated cells) on the accumu- or presence of inhibitors) or subsequently permeabilized lation of [3H]-ATV. E-3-S alone did not alter the CAR upon carrying out the accumulation studies in the absence of [3H]-ATV. However, there was a significant (P≤0.01) or presence of the inhibitors at steady state. As shown decrease in the CAR of [3H]-ATV in cells pretreated with previously (Figure 1C), tariquidar and frusemide did not tariquidar, dipyridamole and frusemide, followed by sub- alter the accumulation of [3H]-ATV, whereas dipyrida- sequent E-3-S coincubation (Figure 2). mole significantly (P<0.01) decreased the accumulation of [3H]-ATV (Figure 2). Permeabilization of the cells with Atazanavir increases the accumulation and decreases digitonin significantly reduced (P<0.01) the accumulation the efflux of daunomycin in the cultured cells of [3H]-ATV (from 15.3 ±0.8 to 13.0 ±0.1). Of the digi- ATV increased the accumulation and decreased the tonin-pretreated cells that were subsequently coincubated efflux of daunomycin in a concentration-dependent with tariquidar or frusemide, we observed no significant manner, with a 50% inhibitory concentration of 9.8 µM decrease in the CAR of [3H]-ATV compared with digi- and 8.5 µM, respectively (Figure 3). However, ATV had tonin-treated samples. By contrast, there was a significant no effects on the accumulation and efflux of daunomy- (P<0.05) decrease in the accumulation of [3H]-ATV in cin in the CEM line (data not shown).

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Figure 3. Concentration-dependent effects of atazanavir on the accumulation and efflux of daunomycin in CEMVBL cells

250

2.5 3 200 2

1.5 150 1

100 0.5 Efflux rate constant (1/s).10 Efflux

Percentage flourescence over control flourescence Percentage 50 0.01 0.1 110 100 Concentration of atazanavir, µM

0 10 20 30 40 50 Concentration of atazanavir, µM

Concentration-dependent effects of atazanavir (0–50 µM) on the accumulation and efflux (tariquidar at 0–200 µM; inset) of daunomycin in CEMVBL cells. Initial efflux rates were determined from the slope of linear regression line fitted to mean fluorescence intensity values determined after 1, 2, 3 and 4 min. Initial daunomycin efflux rates are given as a percentage of the fluorescence values determined for the zero time point. Data points represent the mean of two independently performed experiments. The 50% inhibitory concentration values for the inhibition of efflux (8.5µ M; inset) and enhancement of accumulation (9.8 µM; inset) of daunomycin were calculated from the concentration–response curves using Grafit (Erithacus Software Ltd, Surrey, UK).

Discussion and GF120918) and P-gp/MRP (daidzein) increased the accumulation of [3H]-ATV in CEM and its variant cells. Here, we investigated the contribution of lipophilicity, However, in contrast to the data obtained on other HPIs efflux/influx mechanisms and embedding/sequestration [1,2,13–15,51], the observation that tariquidar, dipyrida- in cell membranes to the accumulation of [3H]-ATV. mole, frusemide, GF120918 and GPV031 all significantly We observed [3H]-ATV to be approximately 7× less decreased or did not alter the accumulation of [3H]-ATV lipophilic than [14C]-LPV, but 1.7× more lipophilic than in some or all of the cells (Figure 1A) was unexpected [3H]-SQV [6]. The octanol/saline partition coefficient (Figure 1A and 1C). Although we found no direct evi- determined for [14C]-mannitol (our positive control) dence that the influx of HPIs are sensitive to these drugs, was similar to that reported previously [49]. and given that none of the drugs (at the concentrations Drug transporters are important determinants of and incubation times employed) are cytotoxic to the cells variable drug accumulation, drug–drug interactions, (data not shown), we postulate that the observed effects efficacy, resistance and toxicity. There is ample evidence might be the result of inhibition of influx, competition that some HPIs are substrates, inhibitors and inducers for accumulation, or displacement of [3H]-ATV from of drug transporters such as P-gp, MRP1/2, BCRP and cell membranes. Indeed, there is evidence that dipyrida- hOATP/hOCTP [1,2,4,7–25,50]. However, the role of mole and frusemide influence the fluxes or exchange of + + 2+ + + - - drug transporters in the accumulation of ATV has not Na , K , Ca , Na /H and Cl /HCO3 [52,53]. Given that been well documented [2,3,25,30,31]. In order to char- E-3-S, a substrate/inhibitor of hOATP/hOCT [4,54], sig- acterize the transport profile of ATV, we studied its accu- nificantly decreased the accumulation of [3H]-ATV, it is mulation in cultured cells and in primary human cells, plausible that the accumulation of [3H]-ATV might be with known transporter expression profiles [1,13]. These linked to the fluxes/exchange of these cations. Despite data provide evidence that ATV is a substrate of P-gp, the recent observation that tariquidar does not inhibit and MRP1. This stems from the observations that there hOATP1B3-mediated uptake of paclitaxel [55], it could was differential accumulation of [3H]-ATV between also be possible that the effects of tariquidar on some CEM and its variant P-gp- and MRP1-overexpressing of the cells might be the result of inhibition of an influx lines and that inhibitors of P-gp/BCRP (that is, tariquidar transporter not described elsewhere.

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As with the cultured CEM and its variant cells, the coincubating tariquidar-, frusemide- and dipyridamole- effects of inhibitors of drug transport proteins on the treated cells with E-3-S significantly decreased the intracellular accumulation of drugs in PBMCs might accumulation of [3H]-ATV (Figure 2). These observa- depend on the relative expression of the transporters. tions indicate the potential for significant drug–drug We recently showed the expression of several drug efflux and drug–endogenous-substance interactions. We also transporters on PBMCs and demonstrated that the accu- observed that ATV enhances both P-gp-mediated accumulation of HPIs is amenable to modulation by inhibi- mulation and inhibits P-gp-mediated efflux of dauno- tors of drug efflux and influx transporters [1,2,6]. Here, mycin in VBL cells (Figure 3). This is in some agree- inhibitors of drug efflux transporters, such as tariquidar, ment with previous studies, which show that although dipyridamole, frusemide and E-3-S, produced contrast- ATV does not induce P-gp or MRP expression [31,61], ing results to that measured for SQV and LPV [1,2]. it is an inhibitor of P-gp (and MRP [2,23,30] and is also The following possibilities might explain these observa- able to increase the plasma concentration of coadminis- tions: reduced cell surface expression of the efflux and tered HPIs [29] via blunting the activities of P-gp/MRP influx transporters; important drug–drug interactions; [2] and possibly cytochrome P450 enzymes [30]. the transporter profile of ATV differs from other HPIs RTV and other HPIs, including ATV, have been – ATV is acid-sensitive to absorption, inhibits hepatic shown to modulate the plasma and intracellular con- uridine 5′-diphospho-glucuronosyltransferase and centrations of other HPIs [2,28,29] via inhibition of induces CYPs [56,57]; and identification of some addi- P-gp/MRP and cytochrome P450. Of the HPIs tested, tional properties of some of the inhibitors used, such only LPV and RTV (at 30 µM) increased the accumula- as frusemide and dipyridamole, might have potential to tion of [3H]-ATV in E1000 cells (Figure 1B), whereas inhibit drug uptake transporters. Clearly, the decrease TPV (at 30 µM) significantly increased accumulation in the accumulation of [3H]-ATV by drugs such as fruse- of [3H]-ATV in PBMCs (Figure 1C). There is evidence mide, dipyridamole and GPV031 (all known inhibitors that some antiretrovirals, such as IDV, RTV, SQV and of efflux transporters) warrants further investigations. efavirenz, are potent inhibitors of uptake transporters Soy-derived phytoestrogens, such as daidzein and gen- (for example, hOATP1B1) or hOCTs [5,62,63]. Some of istein, have been reported to interact with some major the HPIs (for example, APV, IDV and SQV) reduced the drug transporters, including P-gp, MRP, BCRP and accumulation of ATV in the P-gp overexpressing VBL hOATP [42–47,58]. However, their interactions with cells (Figure 1B and 1C). In a previous investigation, we HPIs are largely undefined. We observed that daidzein observed that of the hOATP isoforms studied, only VBL had dual effects, with the agents causing modest but cells express hOATP1A2 [6]. Given that some of these significant decrease (at 1 mM) and increase (at 5 mM) HPIs are also substrates of hOATP [6], it is probably in accumulation of [3H]-ATV (P<0.05) in the cultured that the inhibitory effects of some of the HPIs on the cells. In PBMCs, genistein decreased the accumulation of accumulation of ATV are a direct result of their com- [3H]-ATV, whereas daidzein increased it. The observed petition for entry into the cells with ATV. Overall, these effect of genistein is in agreement with the evidence that observations plus that observed with probenecid suggest it inhibits an influx transporter, showing that it inhibits that ATV might be a substrate of uptake transporters. hOATP1B1-mediated uptake of [3H]-dehydroepiandros- Thus its coadministration with some HPIs or probenecid terone sulfate [45]. Furthermore, it has also been shown might result in alterations in its cellular accumulation that genistein induces the depolarization of membrane Investigations that aim to characterize the role of potential [59], which might ultimately prevent the cellu- drug transporters and the influence of drug embedding/ lar accumulation of [3H]-ATV. As serum concentrations sequestering into cell membranes on the accumulation of phytoestrogens could be high [60], these data suggest of [3H]-ATV in the cells using digitonin produced inter- that intake of soy tablets and or diets rich in soy might esting, but unexpected, findings. This manipulation influence the accumulation, efficacy and toxicity of ATV. decreased CEM-, E1000- and PBMC-associated [3H]- Taken together, these data suggest that these phytoestro- ATV by 47%, 30% and 15%, respectively. However, gens are both inhibitors of influx and efflux transporters a similar manipulation led to a significant increase in and that ATV might be a substrate of transporters that VBL-associated [3H]-ATV, suggesting the possibility that are affected by these phytoestrogens [42–47,58]. [3H]-ATV binds differentially to cells expressing differ- The evidence that E-3-S significantly decreased the ent and varying amounts of transporters, with the drug accumulation of [3H]-ATV in the cultured cells suggests binding more avidly to P-gp- than MRP-overexpressing that ATV, like SQV and LPV [6] is also a substrate of cells. Another possibility is that [3H]-ATV might not be hOATP. Although we observed that E-3-S alone did not efficiently transported by P-gp and MRP. Coincubations decrease the accumulation of [3H]-ATV in the PBMCs containing digitonin-treated cells followed by subse- (Figures 1C and 2), contrary to the lack of effects of quent treatment with tariquidar, frusemide or dipyrida- tariquidar and frusemide alone (Figures 1C and 2), mole were without any additional inhibitory effects over

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Accepted for publication 21 June 2009

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